Objective To evaluate the biocompatibil ity of a new nano TCP/ gelatin / velvet antler polypeptide material. Methods The nano TCP/ gelatin / velvet antler polypeptide material was prepared, and the morphous was observed by scanning electron microscope. L929 and NIH/3T3 cell l ines were cultured conventionally. Acute toxicity test, hemolysis test, cell prol iferation and cytotoxicity test were used to evaluate the biocompatibil ity of the material. Results The compositemicrosphere material was about 10 μm in diamerter and had good spherical geometry, high monodispersity with nanometer size holes on the surface. Toxic symptoms such as hyperspasmia, palsy and death did not appear during the observing stage in acute toxicity test. Maximum hemolysis rate of the material was less than 5% which met the requirement of hemolysis test standard as a medical material. Different concentrations of the materials leaching l iquor could enhance the prol iferation of NIH/3T3 cells, which showed the good biologic activity. Toxicity grade was 0, and the material was no cytotoxic. Conclusion Nano TCP/ gelatin / velvet antler polypeptide material has good biocompatibil ity.
Objective To explore the in vitro osteogenesis of the chitosan-gelatin scaffold compounded with recombinant human bone morphogenetic protein 2 (rhBMP-2). Methods Recombinant human BMP-2 was compounded with chitosan-gelatin scaffolds by freezedrying. 2T3 mouse osteoblasts and C2C12 mouse myoblasts were cultured and seeded onto the complexes at thedensity of 2×104/ml respectively. The complexes were divided into two groups. Group A: 2T3 osteoblasts seeded, consisted of 14 rhBMP-2 modified complexes. Each time three scaffolds were taken on the 3rd, 7th, 14th, and 21st day of the culturing, then the expression of osteocalcin gene (as the marker of bone formation) in adherent cells was detected by semiquantitative RT-PCR with housekeeping gene β-tubulin as internalstandard. The other 2 rhBMP-2 modified complexes were stopped being cultured on 14th day after cell seeding, and the calcification of the complexes was detected by Alizarian Red S staining. Five scaffolds without rhBMP-2 modification as the control group A, they were stopped being cultured on 14th day after cell seeding. Of the 5 scaffolds, 3 were subjected tothe detection of osteocalcin gene expression and 2 were subjected to the detection of calcification. Group B: C2C12 myoblasts seeded, had equal composition andwas treated with the same as group A. Besides these 2 groups, another 2 rhBMP2 modified complexes with 2T3 osteoblasts seeding were cultured for 3 days and then scanned by electron microscope (SEM) as to detect the compatibility of the cell to the complex. ResultsSEM showed that cells attached closely to the complex and grew well. In group A, the expression level(1.28±0.17)of osteocalcin gene in cells on rhBMP-2 modified complexes was higher than that (0.56±0.09) of the control group A, being statistically -significantly different(P＜0.05) control. C2C12 myoblasts which did not express osteocalcin normally could also express osteocalcin after being stimulated by rhBMP-2 for at least 7 days. Alizarian Red S staining showed that there was more calcification on rhBMP-2 modified complexes in both groups. There were more calcification in the group compounded with rhBMP-2, when the groups were seeded with the same cells. Conclusion The complexmade of rhBMP-2 and chitosan-gelatin scaffolds has b osteogenesis ability in vitro.
Objective To investigate the effect of “two-phase” tissue engineered cartilage constructed by autologous marrow mesenchymal stem cells(MSCs) and allogeneic bone matrix gelatin(BMG) in repairing articular cartilage defects. Methods Thirty-twoNew Zealand white rabbits were involved in the experiment. “Two-phase” allogeneic BMG scaffold (one side of porous cancellous bone and the other side of cortical bone; 3 mm both in diameter and in thickness) was prepared from iliac bone and limb bone of 5 rabbits by sequentially chemical method. The MSCs wereseparated from 18 New Zealand white rabbits and induced to express chondrocyticphenotype. The chondrocyte precursor cells were seeded onto “two-phase” allogeneic BMG to construct tissue engineering cartilage. Masson’s trichrome staining, PAS staining and scanning electronic microscopic observation were carried out at 1, 3 and 5 weeks. The defects of full thickness articular cartilage(3 mm both in diameter and in depth) were made at both sides of femoral medial condyles in 27 rabbits(including 18 of separated MSCs and the remaining 9). The defects were repaired with the tissue engineered cartilage at the right side (group A, n=18), with BMG at the left side(group B, n=18), and without any implant at both sides in the remaining 9 rabbits as a control( group C, n=18). After 1, 3 and6 months, the 6 specimens of femoral condyles were harvested in 3 groups, respectively. Gross observation, Masson’s trichrome and Alcian blue staining, modified Wakitani scoring and in situ hybridization of collagen type Ⅱ were carried out to assess the repair efficacy of tissue engineered cartilage. Results The “two-phase” BMG consisted of the dense cortical part and the loose cancellous part. In cancellous part, the pore size ranged 100-800 μm, in which the chondrocyte precursor cells being induced from MSCs proliferated and formed the cell-rich cartilaginous part of tissue engineered cartilage. In cortical part, the pore size ranged 10-40 μm, on which the cells arranged in a layer and formed the hard part of subchondral bone. After 1 month of transplantation, the cartilage and subchondral bone were regenerated in group A; during observation, the regenerated cartilage graduallythinned, but defect was repaired and the structure of the articular surface ansubchondral bone was in integrity. In groups B and C, defects were not repaired, the surrounding cartilage of defect was abrased. According to the modified Wakitani scoring, the indexes in group A were significantly higher than those in group B and C(Plt;0.01) except the thickness of cartilage at 6 months. The positive cell rate of in situ hybridization for collagen type Ⅱ in group A was also higher than those in groups B and C(Plt;0.01). Conclusion “Two-phase” allogeneic BMG is a prospective scaffold for tissue engineered cartilage,which combines with autologous chondrocyte precursor cells induced from MSCs toconstruct the tissue engineering cartilage. The tissue engineered cartilage can repair defects of articular cartilage and subchondral bone.
OBJECTIVE: To prepare chitosan-gelatin/hydroxyapatite (CS-Gel/HA) composite scaffolds, and to investigate the influence of components and preparing conditions to their micromorphology. METHODS: The CS-Gel/HA composite scaffolds were prepared by phase-separation method. Micromorphology and porosity were detected by using scanning electron microscope and liquid displacement method respectively. RESULTS: Porous CS-Gel/HA composite scaffolds could be prepared by phase-separation method, and their density and porosity could be controlled by adjusting components and quenching temperature. CONCLUSION: The study suggests the feasibility of using CS-Gel/HA composite scaffolds for the transplantation of autogenous osteoblasts to regenerate bone tissue.
Objective To evaluate the biomechanicalproperties and structuralcharacteristics of various composites of partially decalcified allogenic bone matrix gelatin and bone cement at different ratios. Methods According to Urist method, partially decalcified allogenic bone matrix gelatin was prepared and mixedwith bone cement at different ratios of 0, 400, 500, and 600mg/g. Then the comparisons of these composites were performed in microstructure, ultimate compression strength and ultimate bending strength properties. Results The electronic microscope showed that the bone particles and bone cement were distributed evenly in the composite, irregularly connecting by multiple points; with the increase ofbone particles and decrease of bone cement in the composite, there were more and more natural crevices, varying from 100 μm to 400 μm in width, in the biomaterials. Of all the composites with the ratios of 0, 400,500, and 600 mg/g, the measurements of ultimate compression strength were (71.7±2.0) MPa, (46.9±3.3) MPa, (39.8±4.1) MPa, and (32.2±3.4) MPa, respectively; and the measurements ofultimate bending strength were (65.0±3.4) MPa, (38.2±4.0) MPa, (33.1±4.3) MPa and (25.3±4.6) MPa, respectively. Conclusion The compositeof partially decalcified allogenic bone matrix gelatin and bone cement has a good biomechanical property and could be easily fabricated and re-shaped, which make it available to be used clinically as an idea bone graft biomaterial.
Although the recent studies have concerned the pathogenesis and therapeutic strategies of acute kidney injury (AKI), the mortality of AKI is still terribly high, and it is still one of the most important death factors in the intensive care unit. There is no doubt that early verdict of AKI, is good for a more aggressive treatment and can promise an improved prognosis for AKI patients. Serum creatinine level, serving as the gold standard for diagnosis of kidney injury, cannot meet current clinical work in its sensitivity and specificity of diagnosis of early AKI. Over the past decades, researchers worked to find and verify novel AKI biomarkers, including neutrophil gelatinase associated lipocalin, interleukin-18, kidney injury molecule-1 and cystatin-C, which were proved to be the potential reliable predictor of AKI development and prognosis, and were of great importance to the early diagnosis and clinical monitoring of AKI. This paper reviews the main studies on these novel prognostic predictors of AKI over the decades and evaluates their roles and limitations in early diagnosis and clinical prognosis prediction.
The purpose of this study is to investigate the effect of superparamagnetic chitosan FGF-2 gelatin microspheres (SPCFGM) on the proliferation and differentiation of mouse mesenchymal stem cells. The superparamagnetic iron oxide chitosan nanoparticles (SPIOCNs) were synthesized by means of chemical co-precipitation, combined with FGF-2. Then The SPCFGM and superparamagnetic chitosan gelatin microspheres (SPCGM) were prepared by means of crosslinking-emulsion. The properties of SPCFGM and SPIONs were measured by laser diffraction particle size analyser and transmisson electron microscopy. The SPCFGM were measured for drug loading capacity, encapsulation efficiency and release pharmaceutical properties in vitro. The C3H10 cells were grouped according to the different ingredients being added to the culture medium: SPCFGM group, SPCGM group and DMEM as control group. Cell apoptosis was analyzed by DAPI staining. The protein expression level of FGF-2 was determined by Western blot. The proliferation activity and cell cycle phase of C3H10 were examined by CCK8 and flow cytometry. The results demonstrated that both of the SPIOCNs and SPCFGM were exhibited structure of spherical crystallization with a diameter of (25±9) nm and (140±12) μm, respectively. There were no apoptosis cells in the three group cells. Both the protein expression level of FGF-2 and cell proliferation activity increased significantly in the SPCFGM group cells(P<0.05). The SPCFGM is successfully constructed and it can controlled-release FGF-2, remained the biological activity of FGF-2, which can promote proliferation activity of C3H10 cells, and are non-toxic to the cell.
Objective To investigate the effect of rhBMP-2 combined with porous CPC on spine fusion in rabbits. Methods rhBMP-2 (1 mg) was loaded with 1 g CPC and 6.0 cm × 2.0 cm × 0.5 cm absorbable gelatin sponge (AGS), respectively, and thereafter frozen to prepare the biomaterial of rhBMP-2/CPC and rhBMP-2/AGS. Forty-five 24-week-old New Zealand rabbits (weight 2.5-3.5 kg) were randomly divided into 3 groups: group A (n=17), group B (n=11) and group C (n=17).With the exposure and removal of L5, 6 transverse process’s posterior bone cortex in all the rabbits, the corresponding cancellous bones were exposed and the posterior bilateral intertransverse bone grafting of L5, 6 were performed on the three groups, then the rhBMP-2/CPC, rhBMP-2/AGS and CPC was implanted into the rabbits of group A, B and C, respectively. Gross observation, histology assay and image examination were conducted 4, 8 and 24 weeks after operation. Results Decalcified hard tissue section demonstrated obvious callus connections in group A, small pieces of callus in group B, and fibrous connection and few cartilage in group C at 4 and 8 weeks after operation. By Kacena measurement standard, the score of group A, B and C at 4 weeks after operation was (7.30 ± 0.76), (3.68 ± 1.60) and (1.75 ± 0.54) points, respectively, and their score at 8 weeks after operation were (8.32 ± 1.11), (3.75 ± 1.23) and (1.47 ± 0.23) points, respectively, indicating there were significant differences between group A and group B as well as between group A and group C at different time points (P lt; 0.05). Undecalcified hard tissue section demonstrated that there was cancellous bone-l ike tissue regeneration in group A, and fiber connection around the implants and l ittle ossification in group C at 4 and 8 weeks after operation. By three dimensions reconstructed CT, group A, B and C scored (2.50 ± 0.57), (1.00 ± 0.00) and (1.00 ± 0.00) points respectively, indicating there was a significant difference between group C and groups A and B as well as between group A and group B (P lt; 0.05). Conclusion As a carrier of rhBMP-2, the CPC is capable of promoting spine bone fusion in rabbits and is a new type of artificial bone repair material.
Abstract To observe the effect of exogenous high molecular weight nerve growth factor (HMW-NGF) mixed with bletilia striata gelatin (BSG) in the promotion of healing, the experiment was performed as follow: (1) In serumfree medium, the normal saline, BSG, HMWNGF, and BSG+HMW-NGF were added separately, and then, the chick embryo root ganglions (DRGs) were cultivated in the above prepared media and the axonal growth was observed. (2) 40 SD rats were divided into 4 groups. A wound of 2cm×2cm was made on the back of every rat. No treatment was given in group one. In other groups, BSG, HMW-NGF, andBSG+HMW-NGF were given separately to the wounds once daily. After 3 and 10 days, the wound area of every rat was measured, cells in the wounds were observed under light microscope and were calculated, and the time of healing was recorded. The results showed that BSG, HMW-NGF, especially BSG+HMW-NGF could promote wound healing.
ObjectiveTo evaluate the biocompatibility of poly lactic acid/bone matrix gelatin (PLA/BMG) composite biomaterial so as to lay a foundation for bone defect repair. MethodsRats'MC3T3-E1 cells were cultured with leaching solution of PLA/BMG and PLA material respectively for 7 days. The cell proliferation rate was tested by MTT and cell toxicity grading was carried out everyday. The PLA/BMG and MC3T3-E1 cells were co-cultured, the cell shape and proliferation were observed by inverted phase contrast microscope at 1, 3, and 5 days and cell adhesion by scanning electron microscope at 5 days. The PLA and PLA/BMG were implanted subcutaneously in 15 Wistar rats. The histological observation was done, and the thickness of fibrous membrane, the number of inflammatory cells, and the vascularization area were measured at postoperative 2nd, 4th, and 8th week. ResultsThe tests for cytotoxicity in vitro showed that the cell proliferation rates were over 100% and the cell cytotoxic grades were grade 0 at 1-7 days in PLA/BMG group. While in PLA group, the cell proliferation rates were less than 100% and the cell cytotoxic grades were grade 1 at 2, 4, and 7 days. After co-culture of PLA/BMG and MC3T3-E1 cells, cells grew on the surface and in the pores of PLA/BMG, and the cellular morphology was triangle or polygon with abundant microvillus on the surface. After subcutaneous implantation, the rats survived to the end of experiment, and incision healed well. PLA was wrapped by connective tissue where there were a lot of lymphocytes and neutrophilic granulocytes. The cells and tissue grew slowly in PLA. The PLA/BMG materials were wrapped by little connective tissue where there were a few inflammatory cells. The connective tissue ingrowth was observed in the center of PLA/BMG. There was no significant difference in the thickness of fibrous membrane between 2 groups at each time point (P>0.05). The number of inflammatory cells of PLA/BMG group were significantly less than those in PLA group at 2, 4, and 8 weeks (P<0.05); the vascularization area was significantly larger than that in PLA group (P<0.05). ConclusionPLA/BMG composite biomaterials prepared by super critical-CO2 technique are good in cell and tissue biocompatibilty.